WO2014134543A1 - Procédés de préparation de tubulysines - Google Patents

Procédés de préparation de tubulysines Download PDF

Info

Publication number
WO2014134543A1
WO2014134543A1 PCT/US2014/019605 US2014019605W WO2014134543A1 WO 2014134543 A1 WO2014134543 A1 WO 2014134543A1 US 2014019605 W US2014019605 W US 2014019605W WO 2014134543 A1 WO2014134543 A1 WO 2014134543A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
formula
optionally substituted
alkyl
mmol
Prior art date
Application number
PCT/US2014/019605
Other languages
English (en)
Inventor
Iontcho Radoslavov Vlahov
Christopher Paul Leamon
Original Assignee
Endocyte, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Endocyte, Inc. filed Critical Endocyte, Inc.
Priority to CA2901337A priority Critical patent/CA2901337A1/fr
Priority to US14/771,532 priority patent/US9771391B2/en
Priority to EP14756463.7A priority patent/EP2961764A4/fr
Publication of WO2014134543A1 publication Critical patent/WO2014134543A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp

Definitions

  • the invention described herein pertains to processes for preparing tubulysins and derivatives thereof.
  • the invention described herein also pertains to processes for preparing tubulysin conjugates.
  • the invention described herein also pertains to processes for preparing intermediates for preparing tubulysin conjugates.
  • Tubulysins are members of a new class of natural products isolated from myxobacterial species (F. Sasse, et al., J. Antibiot. 2000, 53, 879-885). As cytoskeleton interacting agents, the tubulysins are mitotic poisons that inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis (H. Steinmetz, et al., Chem. Int. Ed. 2004, 43, 4888- 4892; M. Khalil, et al., ChemBioChem. 2006, 7, 678-683;G. Kaur, et al., Biochem. J. 2006, 396, 235-242). Tubulysins are extremely potent cytotoxic molecules, exceeding the cell growth inhibition of any clinically relevant traditional chemo therapeutic, including
  • tubulysins are potent against multidrug resistant cell lines (A. Domling, et al., Mol. Diversity 2005, 9, 141-147). These compounds show high cytotoxicity tested against a panel of cancer cell lines with IC 50 values in the low picomolar range; thus, they are of interest as potential anticancer therapeutics.
  • tubulysins and processes for preparing tubulysins are described herein.
  • tubulysins and processes for preparing them are described herein that include linear tetrapeptoid backbones, including illustrative compounds having formula T or TE
  • Ari is optionally substituted aryl or optionally substituted heteroaryl
  • Ri is hydrogen, optionally substituted alkyl, optionally substituted arylalkyl or a pro-drug forming group
  • R 2 is selected from the group consisting of optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted arylalkyl, and optionally substituted heteroarylalkyl;
  • R 12 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted;
  • R 3 is optionally substituted alkyl
  • R 4 is optionally substituted alkyl or optionally substituted cycloalkyl
  • R5 and R 6 are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;
  • R 7 is hydrogen or optionally substituted alkyl
  • n 1, 2, 3, or 4.
  • tubulysin amide and hydrazide derivatives and processes for preparing them are described herein, such as compounds of the following formulae
  • R 1; R 2 , R 12 , R 3 , R4, R5, R 6 , R7, and n are each independently selected from any of the various embodiments described herein; and m is 1 or 2.
  • tubulysins and processes for preparing them are described herein that comprise one or more radicals formed from N-methyl pipecolic acid (Mep), isoleucine (He), and/or one or more non-naturally occurring or hydrophobic amino acid segments such as
  • tubulysins and processes for preparing them are described herein that have formula 1, and are naturally occurring.
  • tubulysin D A total synthesis of tubulysin D has been reported (see, Peltier et al., J Am Chem Soc 128: 16018-19 (2006)). Recently, a modified synthetic protocol toward the synthesis of tubulysin B has been reported (Pando et at., Org Lett 11:5567-69 (2009)).
  • tubulysins for anticancer therapeutics
  • the interest in using tubulysins for anticancer therapeutics accents the need for reliable and efficient processes for preparing tubulysins, and analogs and derivatives thereof. Described herein are improved processes for making tubulysins, or analogs or derivatives thereof, including compounds of formula T and TE.
  • tubulysins or analogs or derivatives thereof, including compounds of formula T and TE, are described.
  • the processes include one or more steps in the following embodiments.
  • a process for preparing a compound of formula B, wherein R5 and R 6 are independently selected from any of the various embodiments described herein, and Rg is CrC 6 n-alkyl or arylalkyl, such as benzyl.
  • the process comprises the step of treating a compound of formula A with a silylating agent, such as triethylsilyl chloride, and a base, such as imidazole in an aprotic solvent.
  • a process for preparing a compound of formula C, wherein R 2 , R 5 , R 6 , and Rg are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of treating a compound of formula B with a base and a compound of the formula C1CH 2 0C(0)R 2 in an aprotic solvent at a temperature below ambient temperature, such as in the range from about -78°C to about 0°C; wherein the molar ratio of the compound of the formula C1CH 2 0C(0)R 2 to the compound of formula B from about 1 to about 1.5.
  • a process for preparing a compound of formula D, wherein n, R 2 , R 5 , R 6 , and Rg are each independently selected from any of the various embodiments described herein, and R 7 is optionally substituted alkyl.
  • the process comprises the steps of
  • a process for preparing a compound of formula FE, wherein n, R 2 , R 5 , R 6 , R 7 , and Rg are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of contacting compound D with an alcohol, R 12 OH, where R 12 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst.
  • the transesterification catalyst is trifluoroacetic acid (TFA).
  • the transesterification catalyst is selected from the group consisting of (Ri 3 ) 8 Sn 4 0 2 (NCS)4, (R 13 ) 2 Sn(OAc) 2 , (Ri 3 ) 2 SnO, (R 13 ) 2 SnCl 2 , (R 13 ) 2 SnS, (R 13 ) 3 SnOH, and (R 1 ) SnOSn(R 1 ) , where R 1 is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted.
  • the transesterification catalyst is(R 13 ) 2 SnO.
  • R 13 is methyl, n-butyl, n-octyl, phenyl, o-MeO-phenyl, p-MeO-phenyl, phenethyl, benzyl, and the like.
  • a process for preparing a compound of formula IE, wherein n, R 5 , R 6 , R 7 , and R 12 are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of contacting compound FE with a metal hydroxide or carbonate.
  • a metal hydroxide or carbonate include LiOH, Li 2 C0 3 , NaOH, Na 2 C0 3 , KOH, K 2 C0 3 , Ca(OH) 2 , CaC0 3 , Mg(OH) 2 , MgC0 3 , and the like.
  • a process for preparing a compound of formula G, wherein n, R 2 , R 5 , R 6 , R7, and Rg are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of treating compound D with a h drolase enzyme.
  • a process for preparing a compound of formula I, wherein n, R 2 , R 5 , R 6 , and R 7 are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of treating the silyl ether of com ound G with a non-basic fluoride containing reagent.
  • a process for preparing a compound of formula GE, wherein n, R 2 , R 5 , R 6 , and R 7 are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of contacting com ound G with an alcohol, R 12 OH,and a transesterification catalyst, as described herein.
  • a process for preparing a compound of formula IE, wherein n, R 5 , R 6 , R 7 , and R 12 are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of treating the silyl ether of compound GE with a non-basic fluoride containing reagent.
  • a process for preparing a compound of formula CE, wherein R 5 , R 12 , R6, and Rg are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of treating a compound of formula B with a base and a compound of the formula YCH 2 OR 12 , where Y is a leaving group such as a halogen selected from chloro, bromo, and iodo, in an aprotic solvent at a temperature below ambient temperature, such as in the range from about -78°C to about 0°C; wherein the molar ratio of the compound of the formula YCH 2 OR 12 to the compound of formula B from about 1 to about 1.3.
  • a process for preparing a compound of formula DE, wherein n, R 12 , R 5 , R 6 , and Rg are each independently selected from any of the various embodiments described herein, and R 7 is optionally substituted alkyl.
  • the process comprises the steps of
  • a process for preparing a compound of formula IE, wherein n, R 12 , R 5 , R6, R7, and Rg are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of treating the silyl ether/ester of compound DE with a base.
  • a process for preparing a compound of formula JE, wherein n, R 12 , R 4 , R 5 , R 6 , and R 7 are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of treating a compound of formula IE with an acylating agent of formula R 4 C(0)X 2 , where X 2 is a leaving group.
  • a process for preparing a tubulysin of formula (TE), wherein n, Ar 1; R 1; R 3 , R ⁇ , R5, R 6 , R 7 , and R 12 are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of forming an active ester intermediate from a compound of formula JE; and reacting the active ester intermediate with a compound of the formula M to give a compound of the formula TE.
  • a process for preparing a tubulysin linker derivative of formula (TE-L-X), wherein n, Ar 1 ; R 1 ; R 3 , R4, R5, R 6 , R 7 , and R 12 are each independently selected from any of the various embodiments described herein;
  • L is selected from the group consisting of
  • R A , R B , and R are each independently selected in each instance from the group consisting of hydrogen and alkyl; or at least two of R A , R B , or R are taken together with the attached carbon atoms to form a carbocyclic ring;
  • RA T represents 0 to 4 substituents selected from the group consisting of amino, or derivatives thereof, hydroxy or derivatives thereof, halo, thio or derivatives thereof, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,
  • heteroarylheteroalkyl nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof;
  • X is an activated sulfur that includes a leaving group that allows formation of a bond between the sulfur and a nucleophile.
  • the process comprises the step of contacting compound T-L-X, with an alcohol,
  • T-L-X Compounds of formula T-L-X are prepared according to the processes described in PCT international application serial No. PCT/US2013/034672, the disclosure of which is incorporated herein by reference.
  • the transesterification catalyst is TFA.
  • X is SS-Ar 2 , where Ar 2 is optionally substituted aryl or optionally substituted heteroaryl.
  • Ar 2 is selected from optionally substituted pyridin-2-yl, pyridin-2-yl, nitrophenyl, and the like.
  • a process for preparing a tubulysin linker derivative of formula (TE-L-X), wherein L, X, n, Ar 1; R 1; R 2 , R 3 , R4, R5, R 6 , R7, and R 12 are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of forming an active ester intermediate KE, where Y is a leaving group, from a compound of formula JE; and reacting the active ester intermediate KE with a compound of the formula M-L-X to give a compound of the formula TE-L-X.
  • each group L, X, n, Ar 1; R 1; R 2 , R 3 , R 4 , R5, R6, R7, and R 12 is independently selected in each instance from any of the various embodiments, genera, subgenera, or lists described herein.
  • ⁇ is optionally substituted aryl.
  • ⁇ is optionally substituted heteroaryl.
  • each of Ar 1; Ar 2 , R 1; R 2 , R 12 , R , R 4 , R5, R 6 , and R 7 may include any necessary or desirable conventional protection groups as appropriate for the reaction conditions in the corresponding process step.
  • a process for preparing a compound of formula B, where the process comprises the step of treating a compound of formula A with triethylsilyl chloride and imidazole in an a rotic solvent.
  • the compound 2 is not purified by
  • a process for preparing a compound of formula C, where the process comprises the step of treating a compound of formula B with from about 1 equivalent to about 1.5 equivalent of base and from about 1 equivalent to about 1.5 equivalent of a compound of the formula C1CH 2 0C(0)R2 in an aprotic solvent at a temperature from about -78°C to about 0°C.
  • the process of any one of the preceding embodiments is described wherein about 1 equivalent to about 1.3 equivalent of a compound of the formula YCH 2 OC(0)R 2 is used, where Y is a leaving group, such as halo selected from chloro, bromo, and iodo.
  • Y is a leaving group, such as halo selected from chloro, bromo, and iodo.
  • the process of any one of the preceding embodiments is described, wherein about 1.2 equivalent of a compound of the formula C1CH 2 0C(0)R 2 is used.
  • the process of any one of the preceding embodiments is described wherein R 2 is n-propyl.
  • R 2 is
  • n is 3.
  • R 7 is methyl.
  • a mixture of compound 3 and the pentafluorophenyl ester of D-N-methyl-pipecolic acid is reduced using H 2 and a palladium-on-charcoal catalyst (Pd/C) to yield compound 4.
  • a process for preparing a compound of formula FE, wherein R 5 and R 6 are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R 2 is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R 7 is optionally substituted alkyl; wherein the process comprises the step of contacting compound D with an alcohol, R 12 OH, where R 12 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst.
  • the transesterification catalyst is trifluoroacetic acid (TFA).
  • the transesterification catalyst is selected from the group consisting of
  • R 1 is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted.
  • transesterification catalyst is( R 13 ) 2 SnO.
  • R 13 is methyl, n-butyl, n- octyl, phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, benzyl, and the like.
  • compound 4 is heated with an alcohol and di-n- butyltin oxide at about 100°C to yield ether FE1 (10), where R is as defined in the various embodiments herein. It is appreciated that a co-solvent may be present.
  • the molar ratio (tin oxide)/(compound 4) is about 0.01 to about 0.30, or about 0.02 to about 0.20, or about 0.05 to about 0.15, or about 0.05 to about 0.10
  • a process for preparing a transesterification product of each of the compounds of formula A and/or B, wherein R5, R 6 , and R 8 are as described in the various embodiments herein, and where Ri 2 is different from Rg; wherein the process comprises the step of contacting compound A or B with an alcohol, R 12 OH, and a transesterification catalyst, as described herein.
  • a process for preparing a compound of formula IE, wherein R5 and R 6 are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R 12 is as described in the various embodiments herein, such as being selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and R 7 is optionally substituted alkyl; wherein the process comprises the step of contacting compound FE with a metal hydroxide or carbonate.
  • Illustrative examples of a metal hydroxide or carbonate include LiOH, Li 2 C0 3 , NaOH, Na 2 C0 3 , KOH, K 2 C0 3 , Ca(OH) 2 , CaC0 3 , Mg(OH) 2 , MgC0 3 , and the like.
  • R5, R 6 , R 7 , and R 12 may each include conventional protection groups on the optional substituents.
  • compound FE1 (10) is treated with LiOH.H 2 0 in a mixture of THF and water at about room temperature to yield compound IE1 (11). It is appreciated that the THF may be replaced with other solvents.
  • a process for preparing a compound of formula JE, wherein R 5 , R 6 , R12, R4, and R 7 are each independently selected from any of the various embodiments described herein; wherein the process comprises the step of treating a compound of formula IE with an acylating agent of formula R 4 C(0)X 2 , where X 2 is a leaving group. It is appreciated that the resulting product may contain varying amounts of the mixed anhydride of compound JE and R 4 C0 2 H.
  • the process described in the preceding embodiment further comprises the step of treating the reaction product with water to prepare JE, free of or substantially free of anhydride.
  • compound IE1 (11) is treated with acetic anhydride in pyridine. It is appreciated that the resulting product JE1 (12) may contain varying amounts of the corresponding mixed anhydride of the terminal carboxylic acid of JE1 (12) and acetic acid.
  • treatment of the reaction product resulting from the preceding step with water in dioxane yields compound JE1 (12), free of or substantially free of the corresponding anhydride. It is to be understood that other solvents can be substituted for dioxane in the hydrolysis of the intermediate mixed anhydride. Alternatively, the step may be performed without solvent.
  • a process for preparing a tubulysin of formula (TE), wherein Ari is optionally substituted aryl; Ri is hydrogen, optionally substituted alkyl, optionally substituted arylalkyl or a pro-drug forming group; R 5 and R 6 are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R 3 is optionally substituted alkyl; R 4 is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R 12 is as described in the various embodiments herein, such as being selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and R 7 is optionally substituted alkyl, alkenyl, alkynyl, hetero
  • a process for preparing a tubulysin linker derivative of formula (TE-L-X), wherein L, X, n, Ar 1 ; R 1 ; R , R ⁇ , R5, R 6 , R 7 , and R 12 are each independently selected from any of the various embodiments described herein.
  • the process comprises the step of forming an active ester intermediate KE, where Y is a leaving group, from a compound of formula JE; and reacting the active ester intermediate KE with a compound of the formula M-L-X to give a compound of the formula TE-L-X.
  • compound JE is treated with an excess amount of active ester forming agent and pentafluorophenol to form the pentafluorophenol ester of compound JE, followed by removal of the excess active ester forming agent prior to the addition of compound I.
  • Ari is phenyl.
  • Ari is substituted phenyl.
  • Ari is 4-substituted phenyl.
  • Ari is RA- substituted phenyl.
  • Ari is 4-hydroxyphenyl, or a hydroxyl protected form thereof.
  • R 3 is methyl.
  • Ri is hydrogen.
  • compound JE1 (12) is treated with an excess amount of a polymeric version of a carbodiimide and pentafluorophenol to form the pentafluorophenyl ester of JE1 (12), the polymeric carbodiimide is removed by filtration; and amino acid (S)- tubutyrosine is added to the solution to yield the tubulysin, compound TE1 (13).
  • the ether analog 8-e can be converted to the ether analog 2-e, via the ether analog 15-e, where R is allyl, or CH 2 (CH 2 ) n CH 3 , and n is 1, 2, 3, 4, 5, or 6.
  • a compound FE is described wherein R 12 , R 5 , R6, and R 7 are as described in the any of the embodiments described herein.
  • R is selected from any of the various embodiments described herein.
  • compound IE 1(11) is described.
  • compound JE1 (12) is described.
  • a compound JE is described wherein R 4 is Me and R 12 , R 5 , R 6 , and R 7 are as described in any of the embodiments described herein.
  • R5 is isopropyl.
  • R 3 is methyl.
  • Ri is hydrogen.
  • acyloxymethyl group (R2-C(0)-0-CH2) present on any of compounds C, D, F, G, H, and T may be converted into the corresponding ether group (R 2-O-CH2), or other group using the process of contacting the compound with trifluoroacetic acid (TFA), as described herein, and also as described in WO 2009/055562, the disclosure of which is incorporated herein by reference.
  • TFA trifluoroacetic acid
  • Ari is optionally substituted aryl or optionally substituted heteroaryl
  • Ri is hydrogen, optionally substituted alkyl, optionally substituted arylalkyl or pro-drug forming group
  • R 2 is selected from the group consisting of optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted arylalkyl, and optionally substituted heteroarylalkyl;
  • R 12 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroarylalkyl, or heteroarylalkyl, each of which is optionally substituted;
  • R 3 is optionally substituted alkyl
  • R 4 is optionally substituted alkyl or optionally substituted cycloalkyl
  • R5 and R 6 are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;
  • R 7 is hydrogen or optionally substituted alkyl
  • n 1, 2, 3, or 4; the process comprising
  • step (a) treating a compound of formula A with triethylsilyl chloride and imidazole in an aprotic solvent, where Rg is CrC 6 unbranched alkyl, such as methyl, or arylalkyl, such as benzyl;
  • Y is a leaving group, such as chloro, bromo, or iodo, and where Rg is
  • C -C unbranched alkyl such as methyl, or arylalkyl, such as benzyl
  • transesterification catalyst selected from the group consisting of trifluoroacetic acid, (R 13 )gSn 4 0 2 (NCS)4, (R 13 ) 2 Sn(OAc) 2 , (Ri 3 ) 2 SnO, (R 13 ) 2 SnCl 2 , (Ri 3 ) 2 SnS, (R 13 ) 3 SnOH, and (R 13 ) 3 SnOSn(R 13 ) 3 , where R 13 is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted;
  • n, Ar 1 ; Ar 2 , R 2 , R 3 , R4, R5, R 6 , R7, and R 12 are each independently selected from any of the embodiments described herein;
  • L is selected from the group consisting of
  • p is an integer from about 1 to about 3
  • m is an integer from about 1 to about 4, and * indicates the points of attachment;
  • R A , R B , and R are each independently selected in each instance from the group consisting of hydrogen and alkyl; or at least two of R A , R B , or R are taken together with the attached carbon atoms to form a carbocyclic ring;
  • RA T represents 0 to 4 substituents selected from the group consisting of amino, or derivatives thereof, hydroxy or derivatives thereof, halo, thio or derivatives thereof, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl,
  • heteroarylheteroalkyl nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof;
  • transesterification catalyst is independently selected from any of the embodiments described herein.
  • n, L, Ar 1; Ar 2 , R 3 , R4, R5, R 6 , R7, and R 12 are each independently selected from any of the embodiments described herein the process comprising the step of mixing a compound of the formula
  • step (b) is performed in an aprotic solvent; and/or step (b) is performed at a temperature from about -78°C to about 0°C; and/or step (b) is performed where the molar ratio of the compound of the formula
  • YCH 2 OC(0)R 2 to the compound of formula B is from about 1 to about 1.5.
  • the transesterification catalyst is selected from the group consisting of (R 13 ) 8 Sn 4 0 2 (NCS)4, (R 13 ) 2 Sn(OAc) 2 , (Ri 3 ) 2 SnO, (Ri 3 ) 2 SnCl 2 , (Ri 3 ) 2 SnS, (Ri 3 ) 3 SnOH, and (R 13 ) 3 SnOSn(R 13 ) 3 , where R 13 is independently selected from any of the embodiments described herein.
  • R 2 , R5, R 6 , Rs, and R 12 are each independently selected from any of the embodiments described herein.
  • transesterification catalyst selected from the group consisting of (R 13 )gSn 4 0 2 (NCS)4, (R 13 ) 2 Sn(OAc) 2 , (Ri 3 ) 2 SnO, (R 13 ) 2 SnCl 2 , (Ri 3 ) 2 SnS, (R 13 ) 3 SnOH, and (R 13 ) 3 SnOSn(R 13 ) 3 , where R 1 and R 12 OH; where R 2 , R5, R 6 , R % , R 12> , and R 1 are each independently selected from any of the embodiments described herein.
  • R 12 OCH 2 X, where X is CI or Br; and R5, R 6 , and R 12 are each
  • R 12 OCH 2 X is n-C 5 H n OCH 2 Br.
  • R 14 is Et 3 Si or RjQO
  • n, R 2 , R 4 , R5, R 6 , R 7 , Rs, and R 12 are each independently selected from any of the embodiments described herein.
  • a transesterification catalyst selected from the group consisting of (R 13 ) 8 Sn 4 0 2 (NCS) 4 , (Ri 3 ) 2 Sn(OAc) 2 , (Ri 3 ) 2 SnO, (Ri 3 ) 2 SnCl 2 , (Ri 3 ) 2 SnS, (Ri 3 ) 3 SnOH, and (R 13 ) 3 SnOSn(R 13 ) 3 , wherein n, R 2 , R 5 , R 6 , R 7 , Rg, Ri 2 , and R 13 are each independently selected from any of the embodiments described herein.
  • n, R 2 , R 3 , R4, R5, R 6 , R7, Ar 1; Ar 2 , L and R 12 are each independently selected from any of the embodiments described herein.
  • the metal carbonate is selected from Li 2 C0 3 , Na 2 C0 3 , K 2 C0 3 , CaC0 3 , or MgC0 3 .
  • the metal hydroxide is selected from LiOH, NaOH, KOH, Ca(OH) 2 , or Mg(OH) 2
  • ⁇ is optionally substituted aryl
  • Ri is hydrogen, alkyl, arylalkyl or a pro-drug forming group
  • R 12 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted;
  • R 3 is optionally substituted alkyl
  • R 4 is optionally substituted alkyl or optionally substituted cycloalkyl
  • R5 and R 6 are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl; R 7 is optionally substituted alkyl; and n is 1, 2, 3, or 4.
  • Ri is hydrogen, alkyl, arylalkyl or a pro-drug forming group
  • R 2 is selected from the group consisting of optionally substituted alkyl, optionally substituted cycloalkyl, and optionally substituted arylalkyl; and/or
  • R 2 is selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl; and/or
  • R 7 is optionally substituted alkyl.
  • R 4 is alkyl
  • Ri is hydrogen, benzyl, or C1-C4 alkyl.
  • R 2 is Ci-C 8 alkyl or C 3 -C 8 cylcoalkyl.
  • R 2 is n-butyl
  • R 3 is methyl
  • R 4 is C1-C8 alkyl or C3-C8 cylcoalkyl.
  • R 4 is methyl
  • R 5 is branched C3-C6 or C3-C8 cycloalkyl.
  • R 6 is branched C3-C6 or C3-C8 cycloalkyl.
  • R 6 is sec-butyl
  • R 7 is C1-C6 alkyl.
  • R 7 is methyl
  • Ari is substituted phenyl.
  • Ari is phenyl. * The process or compound of any one of the preceding clauses wherein Ari is 4-hydroxyphenyl, or a hydroxyl protected form thereof.
  • O- L -S is 0-(CR 2 ) p CR i aa RR bb --SS.
  • R a and R b are each hydrogen.
  • R 8 is methyl
  • n 1, 2, 3, 4, 5, or 6.
  • tubulysin refers both collectively and individually to the naturally occurring tubulysins, and the analogs and derivatives of tubulysins, including the tubulysins prepared using the processes described herein.
  • tubulysin generally refers to the compounds described herein and analogs and derivatives thereof. It is also to be understood that in each of the foregoing, any corresponding pharmaceutically acceptable salt is also included in the illustrative embodiments described herein.
  • derivatives may include prodrugs of the compounds described herein, compounds described herein that include one or more protection or protecting groups, including compounds that are used in the preparation of other compounds described herein.
  • the term "in a protected form” refers to the attachment to the functional group "in a protected form” of any protecting group for that functional group known to a person skilled in the art of organic synthesis.
  • tubulysin also refers to prodrug derivatives of the compounds described herein, and including prodrugs of the various analogs and derivatives thereof.
  • tubulysin refers to both the amorphous as well as any and all morphological forms of each of the compounds described herein.
  • tubulysin refers to any and all hydrates, or other solvates, of the compounds described herein.
  • the compounds described herein may contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers. It is to be understood that in one embodiment, the invention described herein is not limited to any particular stereochemical requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be optically pure, or may be any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. It is also to be understood that such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration at one or more other chiral centers.
  • the compounds described herein may include geometric centers, such as cis, trans, (E)-, and (Z)- double bonds. It is to be understood that in another embodiment, the invention described herein is not limited to any particular geometric isomer requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be pure, or may be any of a variety of geometric isomer mixtures. It is also to be understood that such mixtures of geometric isomers may include a single configuration at one or more double bonds, while including mixtures of geometry at one or more other double bonds.
  • aprotic solvent refers to a solvent which does not yield a proton to the solute(s) under reaction conditions.
  • nonprotic solvents are tetrahydrofuran (THF), 2,5-dimethyl-tetrahydrofuran, 2-methyl-tetrahydrofuran, tetrahydropyran, diethyl ether, t-butyl methyl ether, dimethyl formamide,
  • NMP N-methylpyrrolidinone
  • an equivalent amount of a reagent refers to the theoretical amount of the reagent necessary to transform a starting material into a desired product, i.e. if 1 mole of reagent is theoretically required to transform 1 mole of the starting material into 1 mole of product, then 1 equivalent of the reagent represents 1 mole of the reagent; if X moles of reagent are theoretically required to convert 1 mole of the starting material into 1 mole of product, then 1 equivalent of reagent represents X moles of reagent.
  • active ester forming agent generally refers to any reagent or combinations of reagents that may be used to convert a carboxylic acid into an active ester.
  • active ester generally refers to a carboxylic acid ester compound wherein the divalent oxygen portion of the ester is a leaving group resulting in an ester that is activated for reacting with compounds containing functional groups, such as amines, alcohols or sulfhydryl groups.
  • active ester-forming compounds are N-hydroxysuccinimide, N-hydroxyphthalimide, phenols substituted with electron withdrawing groups, such as but not limited to 4-nitrophenol, pentafluorophenol, ⁇ , ⁇ '-disubstituted isoureas, substituted hydroxyheteroaryls, such as but not limited to 2- pyridinols, 1-hydroxybenzotriazoles, l-hydroxy-7-aza-benzotriazoles, cyanomethanol, and the like.
  • the reaction conditions for displacing the active ester with a compound having an amino, hydroxy or thiol group are mild.
  • reaction conditions for displacing the active ester with a compound having an amino, hydroxy or thiol group are performed at ambient or below ambient temperatures.
  • the reaction conditions for displacing the active ester with a compound having an amino, hydroxy or thiol group are performed without the addition of a strong base.
  • the reaction conditions for displacing the active ester with a compound having an amino, hydroxy or thiol group are performed with the addition of a tertiary amine base, such as a tertiary amine base having a conjugate acid pKa of about 11 or less, about 10.5 or less, and the like.
  • alkyl includes a chain of carbon atoms, which is optionally branched.
  • alkenyl and alkynyl includes a chain of carbon atoms, which is optionally branched, and includes at least one double bond or triple bond, respectively. It is to be understood that alkynyl may also include one or more double bonds. It is to be further understood that in certain embodiments, alkyl is advantageously of limited length, including Q-C ⁇ , C ⁇ -Cn, Q-Cg, C -C , and Ci-C 4 .
  • alkenyl and/or alkynyl may each be advantageously of limited length, including C 2 -C 24 , C 2 -Q 2 , C 2 -C8, C 2 -C6, and C 2 -C 4 . It is appreciated herein that shorter alkyl, alkenyl, and/or alkynyl groups may add less lipophilicity to the compound and accordingly will have different pharmacokinetic behavior.
  • Illustrative alkyl groups are, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2- pentyl, 3-pentyl, neopentyl, hexyl, heptyl, octyl and the like.
  • cycloalkyl includes a chain of carbon atoms, which is optionally branched, where at least a portion of the chain in cyclic. It is to be understood that cycloalkylalkyl is a subset of cycloalkyl. It is to be understood that cycloalkyl may be polycyclic. Illustrative cycloalkyl include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentyleth-2-yl, adamantyl, and the like.
  • cycloalkenyl includes a chain of carbon atoms, which is optionally branched, and includes at least one double bond, where at least a portion of the chain in cyclic. It is to be understood that the one or more double bonds may be in the cyclic portion of cycloalkenyl and/or the non-cyclic portion of cycloalkenyl. It is to be understood that cycloalkenylalkyl and cycloalkylalkenyl are each subsets of cycloalkenyl. It is to be understood that cycloalkyl may be polycyclic.
  • Illustrative cycloalkenyl include, but are not limited to, cyclopentenyl, cyclohexylethen-2-yl, cycloheptenylpropenyl, and the like. It is to be further understood that chain forming cycloalkyl and/or cycloalkenyl is advantageously of limited length, including C 3 -C24, C 3 -Q2, C 3 -C8, C 3 -C 6 , and C5-C 6 . It is appreciated herein that shorter alkyl and/or alkenyl chains forming cycloalkyl and/or cycloalkenyl, respectively, may add less lipophilicity to the compound and accordingly will have different pharmacokinetic behavior.
  • heteroalkyl includes a chain of atoms that includes both carbon and at least one heteroatom, and is optionally branched.
  • Illustrative heteroatoms include nitrogen, oxygen, and sulfur. In certain variations, illustrative heteroatoms also include phosphorus, and selenium.
  • cycloheteroalkyl including heterocyclyl and heterocycle, includes a chain of atoms that includes both carbon and at least one heteroatom, such as heteroalkyl, and is optionally branched, where at least a portion of the chain is cyclic.
  • Illustrative heteroatoms include nitrogen, oxygen, and sulfur. In certain variations, illustrative heteroatoms also include phosphorus, and selenium.
  • Illustrative cycloheteroalkyl include, but are not limited to, tetrahydrofuryl, pyrrolidinyl,
  • aryl includes monocyclic and polycyclic aromatic groups, including aromatic carbocyclic and aromatic heterocyclic groups, each of which may be optionally substituted.
  • carbocyclic and aromatic heterocyclic groups each of which may be optionally substituted.
  • carbocyclic groups each of which may be optionally substituted.
  • Illustrative aromatic carbocyclic groups described herein include, but are not limited to, phenyl, naphthyl, and the like.
  • heteroaryl includes aromatic heterocyclic groups, each of which may be optionally substituted.
  • Illustrative aromatic heterocyclic groups include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl, and the like.
  • amino includes the group NH 2 , alkylamino, and dialkylamino, where the two alkyl groups in dialkylamino may be the same or different, i.e. alkylalkylamino.
  • amino includes methylamino, ethylamino, dimethylamino, methylethylamino, and the like.
  • amino modifies or is modified by another term, such as aminoalkyl, or acylamino the above variations of the term amino are included therein.
  • aminoalkyl includes H 2 N-alkyl, methylaminoalkyl, ethylaminoalkyl, dimethylaminoalkyl, methylethylaminoalkyl, and the like.
  • acylamino includes acylmethylamino, acylethylamino, and the like.
  • amino and derivatives thereof includes amino as described herein, and alkylamino, alkenylamino, alkynylamino, heteroalkylamino,
  • heteroalkenylamino heteroalkynylamino, cycloalkylamino, cycloalkenylamino,
  • cycloheteroalkylamino cycloheteroalkenylamino, arylamino, arylalkylamino
  • amino derivative also includes urea, carbamate, and the like.
  • hydroxy and derivatives thereof includes OH, and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkenyloxy, arylalkynyloxy, acyloxy, and the like, each of which is optionally substituted.
  • hydroxy derivative also includes carbamate, and the like.
  • thio and derivatives thereof includes SH, and alkylthio, alkenylthio, alkynylthio, heteroalkylthio, heteroalkenylthio, heteroalkynylthio, cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio, cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkenylthio, arylalkynylthio, acylthio, and the like, each of which is optionally substituted.
  • thio derivative also includes thiocarbamate, and the like.
  • acyl includes formyl, and alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, heteroalkylcarbonyl, heteroalkenylcarbonyl,
  • heteroalkynylcarbonyl cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl, cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl, arylalkenylcarbonyl,
  • arylalkynylcarbonyl arylalkynylcarbonyl, acylcarbonyl, and the like, each of which is optionally substituted.
  • carboxylate and derivatives thereof includes the group C0 2 H and salts thereof, and esters and amides thereof, and CN.
  • optionally substituted includes the replacement of hydrogen atoms with other functional groups on the radical that is optionally substituted.
  • Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof, and the like.
  • any of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, and/or sulfonic acid is optionally substituted.
  • optionally substituted aryl includes the replacement of hydrogen atoms with other functional groups on the aryl that is optionally substituted.
  • Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof, and the like.
  • any of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, and/or sulfonic acid is optionally substituted.
  • Illustrative substituents include, but are not limited to, a radical -(CH 2 ) X Z , where x is an integer from 0-6 and Z is selected from halogen, hydroxy, alkanoyloxy, including CrC 6 alkanoyloxy, optionally substituted aroyloxy, alkyl, including CrC 6 alkyl, alkoxy, including C -C alkoxy, cycloalkyl, including C 3 -C 8 cycloalkyl, cycloalkoxy, including C 3 -C 8 cycloalkoxy, alkenyl, including C 2 -C 6 alkenyl, alkynyl, including C 2 -C 6 alkynyl, haloalkyl, including CrC 6 haloalkyl, haloalkoxy, including CrC 6 haloalkoxy, halocycloalkyl, including C 3 -C 8 halocycloalkyl, halocycloalkoxy, including
  • alkyl alkylcarbonylamino, aminoalkyl, C -C alkylaminoalkyl, (C -C alkyl)(Ci-C 6
  • Z x is selected from -C0 2 R 4 and -CONR 5 R 6 , where R 4 , R 5 , and R 6 are each independently selected in each occurrence from hydrogen, C -C alkyl, and aryl-Ci-C6 alkyl.
  • prodrug generally refers to any compound that when administered to a biological system generates a biologically active compound as a result of one or more spontaneous chemical reaction(s), enzyme-catalyzed chemical reaction(s), and/or metabolic chemical reaction(s), or a combination thereof.
  • the prodrug is typically acted upon by an enzyme (such as esterases, amidases, phosphatases, and the like), simple biological chemistry, or other process in vivo to liberate or regenerate the more pharmacologically active drug. This activation may occur through the action of an endogenous host enzyme or a non- endogenous enzyme that is administered to the host preceding, following, or during
  • prodrug administration of the prodrug. Additional details of prodrug use are described in U.S. Pat. No. 5,627, 165; and Pathalk et al., Enzymic protecting group techniques in organic synthesis, Stereosel. Biocatal. 775-797 (2000). It is appreciated that the prodrug is advantageously converted to the original drug as soon as the goal, such as targeted delivery, safety, stability, and the like is achieved, followed by the subsequent rapid elimination of the released remains of the group forming the prodrug.
  • Prodrugs may be prepared from the compounds described herein by attaching groups that ultimately cleave in vivo to one or more functional groups present on the compound, such as -OH-, -SH, -C0 2 H, -NR 2 .
  • Illustrative prodrugs include but are not limited to carboxylate esters where the group is alkyl, aryl, aralkyl, acyloxyalkyl,
  • esters include but are not limited to 1-indanyl, N- oxysuccinimide; acyloxyalkyl groups such as acetoxymethyl, pivaloyloxymethyl,
  • alkoxycarbonyloxyalkyl groups such as ethoxycarbonyloxymethyl, a-ethoxycarbonyloxyethyl, ⁇ -ethoxycarbonyloxyethyl, and the like
  • dialkylaminoalkyl groups including di-lower alkylamino alkyl groups, such as
  • Further illustrative prodrugs contain a chemical moiety, such as an amide or phosphorus group functioning to increase solubility and/or stability of the compounds described herein.
  • Further illustrative prodrugs for amino groups include, but are not limited to, (C 3 -C 20 )alkanoyl; halo-(C -C 20 )alkanoyl; (C -C 20 )alkenoyl; (C 4 -C 7 )cycloalkanoyl; (C 3 -C 6 )- cycloalkyl(C 2 -C 1 6)alkanoyl; optionally substituted aroyl, such as unsubstituted aroyl or aroyl substituted by 1 to 3 substituents selected from the group consisting of halogen, cyano, trifluoromethanesulphonyloxy, (Ci-C 3 )alkyl and (C 1 -C 3 )alkoxy, each of which is optionally
  • prodrugs themselves may not possess significant biological activity, but instead undergo one or more spontaneous chemical reaction(s), enzyme-catalyzed chemical reaction(s), and/or metabolic chemical reaction(s), or a
  • prodrug is biologically active. It is also appreciated that prodrugs may often serves to improve drug efficacy or safety through improved oral bioavailability, pharmacodynamic half-life, and the like. Prodrugs also refer to derivatives of the compounds described herein that include groups that simply mask undesirable drug properties or improve drug delivery.
  • one or more compounds described herein may exhibit an undesirable property that is advantageously blocked or minimized may become pharmacological, pharmaceutical, or pharmacokinetic barriers in clinical drug application, such as low oral drug absorption, lack of site specificity, chemical instability, toxicity, and poor patient acceptance (bad taste, odor, pain at injection site, and the like), and others. It is appreciated herein that a prodrug, or other strategy using reversible derivatives, can be useful in the optimization of the clinical application of a drug.
  • the term "treating", "contacting” or “reacting” when referring to a chemical reaction means to add or mix two or more reagents under appropriate conditions to produce the indicated and/or the desired product. It should be appreciated that the reaction which produces the indicated and/or the desired product may not necessarily result directly from the combination of two reagents which were initially added, i.e., there may be one or more intermediates which are produced in the mixture which ultimately leads to the formation of the indicated and/or the desired product.
  • composition generally refers to any product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. It is to be understood that the compositions described herein may be prepared from isolated compounds described herein or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and/or other morphological forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various hydrates and/or solvates of the compounds described herein.
  • compositions that recite compounds described herein are to be understood to include each of, or any combination of, the various morphological forms and/or solvate or hydrate forms of the compounds described herein.
  • compositions may include one or more carriers, diluents, and/or excipients.
  • the compounds described herein, or compositions containing them, may be formulated in a therapeutically effective amount in any conventional dosage forms appropriate for the methods described herein.
  • compositions containing them may be administered by a wide variety of conventional routes for the methods described herein, and in a wide variety of dosage formats, utilizing known procedures (see generally, Remington: The Science and Practice of Pharmacy, (21 st ed., 2005)).
  • reaction was filtered to remove the imidazole HC1 salt, extracted with de-ionized water, and the aqueous phase was back- washed with dichloromethane, the combined organic phase was washed with brine, dried over Na 2 S0 4 , filtered to remove the Na 2 S0 4 , concentrated under reduced pressure, co-evaporated with toluene and dried under high- vacuum overnight to give 6.4 g of crude product 2 (vs 5.9 g of theoretical yield).
  • TES protected dipeptide was dissolved in 38 mL THF (anhydrous, inhibitor-free) and cooled to -45°C and stirred for 15 minutes before adding KHMDS (0.5 M in toluene, 25.5 mL, 12.8 mmol, 1.1 equiv) drop-wise. After the addition of KHMDS was complete, the reaction mixture was stirred at -45°C for 15 minutes, and chloromethyl butyrate (1.8 mL, 1.2 equiv, 14 mmol) was added. The reaction mixture changed from light yellow to a blueish color.
  • reaction was filtered to remove the imidazole HC1 salt, extracted with de-ionized water, and the aqueous phase was back- washed with dichloromethane, the combined organic phase was washed with brine, dried over Na 2 S0 4 , filtered to remove the Na 2 S0 4 , concentrated under reduced pressure, co-evaporated with toluene and dried under high- vacuum overnight to give 12.2 g of product 2.
  • TES protected dipeptide was dissolved in 80 mL THF (anhydrous, inhibitor-free) and cooled to -45°C and stirred for 15 minutes before adding KHMDS (0.5 M in toluene, 50 mL, 25.0 mmol, 1.05 equiv) drop-wise. After the addition of KHMDS was complete, the reaction mixture was stirred at -45°C for 15 minutes, and chloromethyl butyrate (3.6 mL, 1.2 equiv, 28.3 mmol) was added. The reaction mixture changed from light yellow to a blueish color. TLC (20% EtO Ac/petroleum ether) showed the reaction was complete.
  • the flask was shaken under hydrogen (30-35 psi) for 5 hours.
  • the reaction mixture was analyzed by HPLC. The starting material was found to be less than 3%.
  • the mixture was filtered through diatomaceous earth.
  • the diatomaceous earth was extracted with 200 mL ethyl acetate.
  • the filtrate and the ethyl acetate extract were combined and transferred to a separatory funnel and washed with 1% NaHCO 3 /10% NaCl solution (200 mL x 4).
  • the organic layer was isolated and evaporated on a rotary evaporator under reduced pressure.
  • the crude product was dissolved in 40 mL of MeOH/H 2 0 (3: 1).
  • the crude product solution was loaded onto a Biotage C18 column (Flash 65i, 350g, 450mL, 65 x 200 mm) and eluted with buffer A [lOmM NFLOAc/ACN (1: 1)] and B (ACN, acetonitrile).
  • the fractions were collected and organic solvent was removed by evaporating on a rotary evaporator.
  • 100 mL of 10% NaCl solution and 100 mL of methyl tert-butyl ether (MTBE) were added to the flask and the mixture was transferred to a separatory funnel.
  • the organic layer was isolated and dried over anhydrous Na 2 S0 4 , filtered and evaporated on a rotary evaporator to dryness. 2.5g of tripeptide intermediate 4 was obtained (yield 50%).
  • the flask was shaken under hydrogen (30-35 psi) for 5 hours.
  • the reaction mixture was analyzed by HPLC.
  • the reaction was complete.
  • the mixture was filtered through celite.
  • the celite was washed with 500 mL ethyl acetate.
  • the solutions were combined and transferred to a separatory funnel and washed with 1% NaHCO 3 /10% NaCl solution (250 mL x 4).
  • the organic layer was isolated and evaporated on a rotary evaporator under reduced pressure.
  • the crude product was dissolved in dichloromethane and the urea was filtered.
  • the ether analog of compound 3 can also be prepared. Reductive condensation of that ether analog with MEP yields compound 10 directly.
  • R allyl, alkyl, including CH 2 (CH 2 ) n CH 3 , where n isO, 1, 2, 3, 4, 5, or 6. Also described herein, is the conversion of 4 to 10 (R remains Me) by contacting 4 with TFA and an alcohol. In some illustrative examples of compound 10, R is allyl, or CH 2 (CH 2 )nCH 3 , where n is 1, 2, 3, 4, 5, or 6.
  • the reaction mixture was stirred at ambient temperature for 3 minutes, concentrated on a Buchi Rotavapor at 30°C for 10 minutes, residue stirred at ambient temperature under high vacuum for 75 minutes, and to which was added saturated NaHC0 3 solution (10 mL) with vigorous stirring, followed by addition of acetonitrile (ACN, 3.0 mL).
  • ACN acetonitrile
  • the resulting white suspension was stirred at ambient temperature for 3 minutes and let stand to settle.
  • the top clear solution was loaded onto a Biotage SNAP 12 g KP-C18-HS column on a Biotage system.
  • the white solid left in the reaction flask was dissolved in water (5.0 mL) and the solution was also loaded onto the Biotage column.
  • Step 2 EC1426 (114 mg) was dissolved in anhydrous DCM (1.5 mL) and to which was added TFA (0.50 mL). The resulting solution was stirred at ambient temperature under argon for 70 minutes and concentrated under reduced pressure to give a residue, which was co-evaporated with anhydrous DCM (2.0 mL x 3) and vacuumed at ambient temperature for 9 hours prior to use in Step 3.
  • Step 3 The residue from Step 1 was dissolved in anhydrous DCM (1.5 mL) and to this solution was added DIPEA (0.50 mL) followed by a solution of the residue from Step 2 dissolved in anhydrous dimethylformamide (DMF, 1.5 mL). The resulting solution was stirred at ambient temperature under argon for 1 hour, diluted with ethyl acetate (EtOAc, 60 mL), and washed with brine (20 mL x 3).
  • EtOAc ethyl acetate
  • EXAMPLE General Synthesis of Disulfide Containing Tubulysin Conjugates.
  • a binding ligand-linker intermediate containing a thiol group is taken in deionized water (ca. 20 mg/mL, bubbled with argon for 10 minutes prior to use) and the pH of the suspension was adjusted with aqueous phosphate (bubbled with argon for 10 minutes prior to use) to a pH of about 7.0 (the suspension may become a solution when the pH increased).
  • Additional deionized water is added (ca. 20-25%) to the solution as needed, and to the aqueous solution is added immediately a solution of compound (2) in acetonitrile (ca. 20 mg/mL). The reaction mixture becomes homogenous quickly.
  • the reaction mixture is diluted with 2.0 mM sodium phosphate buffer (pH 7.0, ca 150 volume percent) and the acetonitrile is removed under vacuum. The resulting suspension is filtered and the filtrate may be purified by preparative HPLC. Fractions are lyophilized to isolate the conjugates.
  • the foregoing method is equally applicable for preparing other tubulysin conjugates by the appropriate selection of the tubulysin starting compound, including tubulysin starting compounds having a 3-nitropyridin-2-ylthio activating group.
  • Illustrative binding ligand-linker intermediates are described in WO 2008/112873, the disclosure of which is incorporated herein by reference.
  • This binding ligand-linker intermediate was prepared by SPPS according to the general peptide synthesis procedure described herein starting from H-Cys(4- methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS reagents:
  • Coupling steps In a peptide synthesis vessel add the resin, add the amino acid solution, DIPEA, and PyBOP. Bubble argon for lhr. and wash 3X with DMF and IPA. Use 20% piperidine in DMF for Fmoc deprotection, 3X (lOmin), before each amino acid coupling. Continue to complete all 9 coupling steps. At the end treat the resin with 2% hydrazine in DMF 3X (5min) to cleave TFA protecting group on Pteroic acid, wash the resin with DMF (3X), IPA (3X), MeOH (3X), and bubble the resin with argon for 30 min.
  • Cleavage step Reagent: 92.5% TFA, 2.5% H 2 0, 2.5% triisopropylsilane, 2.5% ethanedithiol.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

Cette invention concerne des procédés de préparation de tubulysines et de dérivés de celles-ci. Cette invention concerne également des procédés de préparation de conjugués de tubulysines ainsi que des procédés de préparation d'intermédiaires pour obtenir lesdits conjugués de tubulysines. Dans une étape, l'alcool alpha-thiazolylique est protégé avec un chlorure de triéthylsilyle en présence d'imidazole dans un solvant aprotique.
PCT/US2014/019605 2013-03-01 2014-02-28 Procédés de préparation de tubulysines WO2014134543A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA2901337A CA2901337A1 (fr) 2013-03-01 2014-02-28 Procedes de preparation de tubulysines
US14/771,532 US9771391B2 (en) 2013-03-01 2014-02-28 Process for preparing tubulysins
EP14756463.7A EP2961764A4 (fr) 2013-03-01 2014-02-28 Procédés de préparation de tubulysines

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201361771429P 2013-03-01 2013-03-01
US61/771,429 2013-03-01
US201361793082P 2013-03-15 2013-03-15
US61/793,082 2013-03-15

Publications (1)

Publication Number Publication Date
WO2014134543A1 true WO2014134543A1 (fr) 2014-09-04

Family

ID=51421261

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/019605 WO2014134543A1 (fr) 2013-03-01 2014-02-28 Procédés de préparation de tubulysines

Country Status (4)

Country Link
US (2) US20140249315A1 (fr)
EP (1) EP2961764A4 (fr)
CA (1) CA2901337A1 (fr)
WO (1) WO2014134543A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2919774A4 (fr) * 2012-11-15 2016-11-09 Endocyte Inc Conjugués pour traiter les maladies provoquées par des cellules exprimant psma
US9505747B2 (en) 2012-03-29 2016-11-29 Endocyte, Inc. Processes for preparing tubulysin derivatives and conjugates thereof
US9951324B2 (en) 2010-02-25 2018-04-24 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
EP3223860A4 (fr) * 2014-11-25 2018-08-01 Endocyte, Inc. Procédés de traitement du cancer par ciblage de macrophages associés à une tumeur
US10046054B2 (en) 2007-08-17 2018-08-14 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10188759B2 (en) 2015-01-07 2019-01-29 Endocyte, Inc. Conjugates for imaging
JP2019515880A (ja) * 2016-03-16 2019-06-13 エンドサイト・インコーポレイテッドＥｎｄｏｃｙｔｅ， Ｉｎｃ． 炭酸無水酵素ｉｘ阻害剤抱合体およびその使用
US10398791B2 (en) 2013-10-18 2019-09-03 Deutsches Krebsforschungszentrum Labeled inhibitors of prostate specific membrane antigen (PSMA), their use as imaging agents and pharmaceutical agents for the treatment of prostate cancer
US11229708B2 (en) 2015-12-04 2022-01-25 Seagen Inc. Conjugates of quaternized tubulysin compounds
US11793880B2 (en) 2015-12-04 2023-10-24 Seagen Inc. Conjugates of quaternized tubulysin compounds

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101098854B (zh) 2004-07-23 2012-12-05 恩多塞特公司 二价连接体及其轭合物
US9555139B2 (en) 2007-03-14 2017-01-31 Endocyte, Inc. Binding ligand linked drug delivery conjugates of tubulysins
EP3569251A1 (fr) 2007-06-25 2019-11-20 Endocyte, Inc. Conjugués contenant des lieurs espaceurs hydrophiles
US9877965B2 (en) 2007-06-25 2018-01-30 Endocyte, Inc. Vitamin receptor drug delivery conjugates for treating inflammation
US10080805B2 (en) 2012-02-24 2018-09-25 Purdue Research Foundation Cholecystokinin B receptor targeting for imaging and therapy
WO2014062697A2 (fr) 2012-10-16 2014-04-24 Endocyte, Inc. Conjugués d'administration de médicament contenant des acides aminés artificiels et procédés d'utilisation
WO2017031209A1 (fr) * 2015-08-18 2017-02-23 Endocyte, Inc. Analogues de tubulysine et procédés associés
AU2017234676A1 (en) 2016-03-16 2018-09-20 Purdue Research Foundation Carbonic anhydrase IX targeting agents and methods

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100240701A1 (en) * 2007-10-25 2010-09-23 Endocyte, Inc. Tubulysins and processes for preparing
US20110027274A1 (en) * 2009-08-03 2011-02-03 Medarex, Inc. Antiproliferative compounds, conjugates thereof, methods therefor, and uses thereof
US20110263650A1 (en) * 2007-07-20 2011-10-27 Helmholtz-Zentrum Für Infektions-Forschung Gmbh Tubulysin D Analogues
WO2012019123A1 (fr) * 2010-08-06 2012-02-09 Endocyte, Inc. Procédés de préparation de tubulysines
WO2013149185A1 (fr) * 2012-03-29 2013-10-03 Endocyte, Inc. Procédés de préparation de dérivés de tubulysine et conjugués de ceux-ci

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5627165A (en) 1990-06-13 1997-05-06 Drug Innovation & Design, Inc. Phosphorous prodrugs and therapeutic delivery systems using same
EP1523493B1 (fr) 2002-07-09 2013-09-04 Dömling, Alexander Nouveaux analogues de tubulysine
AU2003253048A1 (en) 2002-07-09 2004-01-23 Morphochem Aktiengellschaft Fur Kombinatorische Chemie Tubulysin conjugates
DE10254439A1 (de) 2002-11-21 2004-06-03 GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) Tubulysine, Herstellungsverfahren und Tubulysin-Mittel
WO2004069159A2 (fr) 2003-01-27 2004-08-19 Endocyte, Inc. Conjugues de delivrance de medicaments de liaison au recepteur de vitamines
US9555139B2 (en) 2007-03-14 2017-01-31 Endocyte, Inc. Binding ligand linked drug delivery conjugates of tubulysins
DE102009032972A1 (de) 2009-07-14 2011-01-20 Erbe Elektromedizin Gmbh Vorrichtung zur Herstellung von Anastomosen
WO2011069116A1 (fr) 2009-12-04 2011-06-09 Endocyte, Inc. Conjugués de tubulysines d'apport de médicament liés à un ligand de liaison

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110263650A1 (en) * 2007-07-20 2011-10-27 Helmholtz-Zentrum Für Infektions-Forschung Gmbh Tubulysin D Analogues
US20100240701A1 (en) * 2007-10-25 2010-09-23 Endocyte, Inc. Tubulysins and processes for preparing
US20110027274A1 (en) * 2009-08-03 2011-02-03 Medarex, Inc. Antiproliferative compounds, conjugates thereof, methods therefor, and uses thereof
WO2012019123A1 (fr) * 2010-08-06 2012-02-09 Endocyte, Inc. Procédés de préparation de tubulysines
WO2013149185A1 (fr) * 2012-03-29 2013-10-03 Endocyte, Inc. Procédés de préparation de dérivés de tubulysine et conjugués de ceux-ci

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP2961764A4 *

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11083710B2 (en) 2007-08-17 2021-08-10 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US11369590B2 (en) 2007-08-17 2022-06-28 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10646581B2 (en) 2007-08-17 2020-05-12 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10624971B2 (en) 2007-08-17 2020-04-21 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US11298341B2 (en) 2007-08-17 2022-04-12 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10624969B2 (en) 2007-08-17 2020-04-21 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10046054B2 (en) 2007-08-17 2018-08-14 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10624970B2 (en) 2007-08-17 2020-04-21 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US11717514B2 (en) 2007-08-17 2023-08-08 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10828282B2 (en) 2007-08-17 2020-11-10 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10406240B2 (en) 2007-08-17 2019-09-10 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US11318121B2 (en) 2007-08-17 2022-05-03 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10485878B2 (en) 2007-08-17 2019-11-26 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10517957B2 (en) 2007-08-17 2019-12-31 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10517956B2 (en) 2007-08-17 2019-12-31 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US10557128B2 (en) 2010-02-25 2020-02-11 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US9951324B2 (en) 2010-02-25 2018-04-24 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US11155800B2 (en) 2010-02-25 2021-10-26 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US12091693B2 (en) 2010-02-25 2024-09-17 Purdue Research Foundation PSMA binding ligand-linker conjugates and methods for using
US9505747B2 (en) 2012-03-29 2016-11-29 Endocyte, Inc. Processes for preparing tubulysin derivatives and conjugates thereof
US9782493B2 (en) 2012-11-15 2017-10-10 Endocyte, Inc. Conjugates for treating diseases caused by PSMA expressing cells
EP2919774A4 (fr) * 2012-11-15 2016-11-09 Endocyte Inc Conjugués pour traiter les maladies provoquées par des cellules exprimant psma
US10912840B2 (en) 2012-11-15 2021-02-09 Endocyte, Inc. Conjugates for treating diseases caused by PSMA expressing cells
US9636413B2 (en) 2012-11-15 2017-05-02 Endocyte, Inc. Conjugates for treating diseases caused by PSMA expressing cells
US11951190B2 (en) 2013-10-18 2024-04-09 Novartis Ag Use of labeled inhibitors of prostate specific membrane antigen (PSMA), as agents for the treatment of prostate cancer
US10471160B2 (en) 2013-10-18 2019-11-12 Deutsches Krebsforschungszentrum Labeled inhibitors of prostate specific membrane antigen (PSMA), their use as imaging agents and pharmaceutical agents for the treatment of prostate cancer
US10398791B2 (en) 2013-10-18 2019-09-03 Deutsches Krebsforschungszentrum Labeled inhibitors of prostate specific membrane antigen (PSMA), their use as imaging agents and pharmaceutical agents for the treatment of prostate cancer
US11931430B2 (en) 2013-10-18 2024-03-19 Novartis Ag Labeled inhibitors of prostate specific membrane antigen (PSMA) as agents for the treatment of prostate cancer
US11045564B2 (en) 2013-10-18 2021-06-29 Deutsches Krebsforschungszentrum Labeled inhibitors of prostate specific membrane antigen (PSMA) as agents for the treatment of prostate cancer
EP3223860A4 (fr) * 2014-11-25 2018-08-01 Endocyte, Inc. Procédés de traitement du cancer par ciblage de macrophages associés à une tumeur
US10898596B2 (en) 2015-01-07 2021-01-26 Endocyte, Inc. Conjugates for imaging
US10188759B2 (en) 2015-01-07 2019-01-29 Endocyte, Inc. Conjugates for imaging
US11229708B2 (en) 2015-12-04 2022-01-25 Seagen Inc. Conjugates of quaternized tubulysin compounds
US11793880B2 (en) 2015-12-04 2023-10-24 Seagen Inc. Conjugates of quaternized tubulysin compounds
JP2019515880A (ja) * 2016-03-16 2019-06-13 エンドサイト・インコーポレイテッドＥｎｄｏｃｙｔｅ， Ｉｎｃ． 炭酸無水酵素ｉｘ阻害剤抱合体およびその使用

Also Published As

Publication number Publication date
CA2901337A1 (fr) 2014-09-04
US20140249315A1 (en) 2014-09-04
US9771391B2 (en) 2017-09-26
EP2961764A1 (fr) 2016-01-06
EP2961764A4 (fr) 2016-11-23
US20160016993A1 (en) 2016-01-21

Similar Documents

Publication Publication Date Title
WO2014134543A1 (fr) Procédés de préparation de tubulysines
EP2831059B1 (fr) Procédés de préparation de dérivés de tubulysine et conjugués de ceux-ci
US9499849B2 (en) Processes for preparing tubulysins
JPH0820597A (ja) トロンビン阻害作用を有する複素環カルボニル化合物
KR101223158B1 (ko) 6,11-다리걸친 바이아릴 매크로라이드
AU2021201355A1 (en) Seco macrolide compounds
DE69610145T2 (de) Benzo-kondensierte Azepinon-und Piperidinonverbindungen, nützlich als ACE-und NEP-Inhibitoren
CN110325527B (zh) 作为细胞外柠檬酸盐摄取的抑制剂的磺酰胺
CA2212437A1 (fr) Derives basiques oxazoline-amides des antibiotiques ge 2270 ou similaires
WO2021026066A1 (fr) Compositions de trofinétide
Schmidt et al. The Total Synthesis of Antrimycin Dv; II: Synthesis of Tetrahydropyridazinecarboxylic Acid and Its Incorporation into Peptides
AU2013203491A1 (en) Processes for preparing tubulysins
RU2304583C1 (ru) Способ синтеза ди- и триаминохлоринов
KR100450607B1 (ko) 가라민 유도체의 제조방법
WO2021165927A1 (fr) 2-cyano-pyrrolidines, -pipéridines ou -diazépines utilisées comme agents hyperglycémiques
JPH08109180A (ja) Ｏ→ｎ分子内アシル転位型プロドラッグ
JPH09263577A (ja) ジフェニルメチル−アゼチジノン化合物及びエラスターゼ阻害剤

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14756463

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2901337

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 14771532

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2014756463

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2014756463

Country of ref document: EP